Method for increasing tissue oxygenation

ABSTRACT

Disclosed are methods and compositions for increasing tissue oxygen levels by administration of superoxygenated compositions of tissue surfaces. The methods are applicable to treatment of a wide variety of conditions including burns, bedsores, ulcers, necrosis and anaerobic infections.

This application claims the priority of U.S. patent application Ser. No.10/052,075 filed Jan. 18, 2002 and of U.S. provisional patentapplication Ser. No. 60/265,819 filed Feb. 1, 2001, the entire contentsof each are hereby incorporated by reference.

1.0 BACKGROUND ART

1.1 Field of the Invention

The present invention is directed to oxygenating compositions andmethods for administering high levels of oxygen to subcutaneous andsubepithelial tissues. In particular, methods for surface delivery ofsuper oxygenating compositions for such treatment are described.

1.2 Description of Related Art

In many medical conditions including diabetes, burns, bedsores, andwounds the ability to oxygenate tissue is compromised and arterialoxygen may not reach damaged skin. Tens of thousands of patients dieeach year in the U.S. as a result of complications from insufficientdelivery of oxygen to compromised tissue. Poor oxygen delivery,particularly in the limbs, results in slow healing, infections, scardevelopment, and in the worst cases, tissue death and amputation.

The effect of oxygen tension on wound healing has been extensivelystudied. (For a review, see Whitney, J. D. (1989)). Wound healing isdependent upon several processes including proliferation of fibroblasts,collagen synthesis, angiogenesis and re-epithelialization. Animalstudies have shown that several of these processes are affected by thesubcutaneous partial pressure of oxygen (pO₂). For example, supplementaloxygen can lead to increased rate of collagen deposition,epithelialization and improved healing of split thickness grafts.Increased subcutaneous pO₂ has also been shown to improve bacterialdefenses.

Many skin sores, ulcers, wounds and burns do not heal properly becausethere is a severe depletion of oxygen reaching these affected areas dueto deterioration of the associated blood microcirculation.Conventionally, many of these skin diseases have been treated by variousmethods of administration of oxygen gas, either through inhalation ofthe gas, or by topical treatment with the gas.

The oldest method of administering oxygen gas to a patient is byhyperbaric chamber technology. This is a systemic treatment, involvingplacement of a patient in a closed pressurized chamber. Inside thechamber, the patient breathes elevated levels of oxygen gas. The extraoxygen taken in by inhalation becomes dissolved in the bloodstream anddiffuses into the body tissues, thereby raising the local tissue oxygenlevels. Unfortunately, hyperbaric treatment has not been successful inall situations, in particular where trauma or disease restrict bloodflow to the affected tissue. Treatment of skin diseases by placing apatient in a hyperbaric chamber is costly and time-consuming and manypatients react unfavorably when placed in hyperbaric chambers. Treatmentof many conditions, such as bedsores, for much longer than four hours atone time may induce oxygen toxemia and hence be counterproductive. Toxiceffects of hyperbaric treatment include twitching, ringing in the ears,dizziness, and in some cases severe effects such as coma andconvulsions. Additionally, hyperbaric treatment is expensive and onlyavailable in treatment facilities that are properly equipped withhyperbaric chambers. Patients are only given oxygen through the lungs.The atmosphere of a multichamber hyperbaric unit is ordinary atmosphericgas as there is little known therapeutic value assigned to topicalapplication of oxygen.

To overcome drawbacks associated with systemic hyperbaric treatment,attempts have been made to use “topical hyperbaric” oxygenation devicesdesigned for regional use on an isolated body part such as a limb. Insuch devices, the delivery route for the pressurized oxygen is topical,as opposed to systemic. Only the affected body part is exposed to thepressurized oxygen. Thus the oxygen gas must diffuse from the surface ofthe skin to the underlying tissues. For example, U.S. Pat. No. 4,801,291discloses a portable topical hyperbaric apparatus having a gasimpermeable internal chamber into which therapeutic gases are introducedto treat a portion of the patient's body. Similarly, U.S. Pat. No.5,020,579 discloses a hyperbaric oxygenation apparatus in which a limbis isolated in a portable chamber in the form of an inflatable bag intowhich oxygen gas is administered through an oxygen port in communicationwith a patient respirator connected to an oxygen source. The pressure ofthe oxygen in the collapsible bag is pulsated between maximum andminimum positive values. The patient cyclically experiences first anincrease in the blood gas levels on the limb under treatment with acorresponding restriction in blood flow and, thereafter, a progressivereturn to normal blood flow rates in the limb as the pressure in thechamber changes from maximum to minimum positive pressure.

Several disadvantages exist with the approach of using “topicalhyperbaric” oxygenation devices. For example, an external oxygen sourceand a respirator normally used for respiratory therapy must be suppliedwith the apparatus. In addition, intermittent restriction and release ofblood flow to the treatment area may not be advisable or tolerable foralready compromised tissues.

Alternative topical methods to “topical hyperbaric” treatments forpoorly healing skin lesions involve the topical application of highlevels of oxygen gas through wound dressings. U.S. Pat. No. 5,792,090,discloses an oxygen generating wound dressing and a method of increasingoxygen tension in surface wounds through the application of such abandage. In this method, the wound dressing contains an oxygen permeablemembrane and a reservoir capable of supplying oxygen through a chemicalreaction. U.S. Pat. No. 5,855,570 describes another type ofoxygen-producing bandage to promote healing of skin wounds. This devicecombines a wound dressing with an electrochemical, chemical, or thermalmeans of generating high purity oxygen, and can be regulated to supplyoxygen gas to an area above the wound at various concentrations,pressures and dosages.

Unfortunately, topical treatments with oxygen gas such as by topicalhyperbaric oxygenation and use of oxygen bandages have provided onlyminor improvements in promoting healing of skin disorders and intreating diseases. Moreover, peroxide application can generate singletoxygen O₂ and is a potential source of free radical damage to the skin(Elden, 1995).

1.3 Deficiencies in the Prior Art

Administration of elevated levels of systemic oxygen gas has beenrecognized as beneficial in the treatment of several skin disorders;however, the available delivery methods, such as hyperbaric chambertherapy, topical application of oxygen gas, topical hyperbaric treatmentof isolated limbs and use of oxygen-producing bandages are at bestminimally effective and often lead to problems that include toxicity andpoor oxygen penetration of the skin. Currently used procedures fortreatment of skin disorders such as ulcers, bedsores, and burns mayexacerbate the existing skin disorder.

It is therefore desirable to provide methods of treatment for skindisorders that increase tissue oxygenation to induce more rapid healingof the skin, while not exacerbating an existing condition or causingadditional side effects.

2.0 SUMMARY OF THE INVENTION

Conventional methods of increasing tissue oxygenation employ oxygen gas.In distinct contrast, the present invention discloses a novel method ofincreasing tissue oxygenation by topical application of asuperoxygenated composition. The superoxygenated compositions rapidlyraise oxygen partial pressure levels in the tissue by promotingefficient diffusion of oxygen into the tissue.

Accordingly, the invention discloses a method of increasing tissueoxygenation in mammals, comprising applying a superoxygenatedcomposition to a tissue surface for a time sufficient to increase thesubepithelial partial oxygen pressure from about 30% to about 120% abovebaseline pO₂. The mammal will generally be a human, but there is nolimitation to its use in veterinary applications to small and largeanimals that may have tissue damage responsive to therapeutic proceduresthat increase oxygenation of tissues.

The most common applications are direct application to the external skinbut the method is equally applicable to mucous membrane surfaces of thealimentary canal as well as organ surfaces. Organs may be exposed oractually removed from the body cavity during surgical procedures. Onemay immerse an organ in a superoxygenated solution prepared inaccordance with the invention, contact part of the organ with such apreparation, or perfuse the organ with the superoxygenated solution. Inthe latter case, this may be an ex vivo procedure intended to maintainorgan viability and reduce ischemic damage.

One may desire to increase the oxygen level in tissues for severalreasons, mainly in situations where the tissue is affected by acondition or disease such as bedsores, wounds, burns or ulcers or anycondition that tends to decrease normal tissue oxygen levels.Additionally, It is expected to be particularly beneficial in treatinganaerobic bacterial infections such as those caused by Pseudomonasspecies, Bacteroides species such as Bacteroides fragilis, Prevotellamelaminogenica, Prevotella bivia, Prevotella disiens, Fusobacterium,Actinomyces, Lactobacillus, Propionibacterium, Eubacterium,Bifidobacterium, Arachnia, Peptostreptococcus, Veillonella, Clostridiumspecies such as C. tetani, C. botulinum, C. perfringens, C. difficileand Porphyromonas. These infections may fester internally in lungtissue, oral or vaginal mucosa or become embedded in the surface oforgans such as liver, kidney and heart.

Accordingly, one of the benefits of using the disclosed methods toenhance tissue oxygen levels is the toxicity to pathogenic anaerobicbacteria. A particularly desirable application is to control or kill theanaerobic bacteria responsible for peridontal disease. A superoxygenatedmouthwash solution would be safe and convenient for use and can bepackaged to maintain stability of the superoxygenated solution by usinga pressurized container with means for single dose dispensing orpackaged for single use.

The superoxygenated compositions of the present invention comprise atleast about 55 ppm oxygen but find useful concentrations from about 45to about 220 ppm. The oxygen level in the compositions depends onseveral factors, including the type of composition, the temperature, andother components, active or not, that may be added for various reasonssuch as stability, ease of application or to enhance absorption.

It is well known that gas concentration in fluids will be inverselyproportional to the temperature. When desiring to use aqueous basedsuperoxygenated compositions, the temperature will be dictated not bychemical considerations but by the potential damage to living tissue andby the need for higher oxygen concentrations. Accordingly, where thecompositions are applied locally to external skin surfaces; for exampleto a forearm lesion, solution temperatures of about 0° C. will generallybe considered appropriate. This will provide relatively high oxygenlevels, typically in the range of 220 ppm. On the other hand, a patientmay be whole-body immersed in a whirlpool bath at a more comfortabletemperature in the range of about 34° C. The oxygen concentration willnecessarily be less than 220 ppm due not only to temperature but also tothe open environment commonly used in whirlpool baths in suchestablishments as rehabilitation centers.

The superoxygenated solutions and compositions of the present inventioncomprise oxygen microbubbles. Conventionally pressurized liquids such ascarbonated beverages contain relatively large gas bubbles that escapefairly quickly into the atmosphere once pressure is released. Themicrobubbles employed in the disclosed compositions are much smaller,remain in solution longer and are thus more stable. Importantly, theoxygen provided by the microbubbles is at a partial pressure effectiveto quickly raise subepithelial oxygen partial pressure significantlyabove baseline or normal oxygen partial pressure levels.

As generated for use in the disclosed superoxygenated compositions,microbubble size is typically in the 1-2 micron range. The small size isbelieved to be an important contributor to the beneficial effects oftopical application of solutions containing the microbubbles. The mostpreferred solutions appear to be those in which the oxygen bubbles areno larger than about 8 microns in size; however, a range of microbubblesizes exist in the prepared solutions, at least as small as 0.6 micronsas detected at the limit of resolution by impedence methods for whichresults are illustrated graphically in FIG. 3. A practical range formany applications is between about 1μ and about 10μ in diameter orbetween about 3μ and about 8μ in diameter.

While the microbubble compositions need not be purely aqueous,compositions will normally comprise an aqueous base such as a buffer, ora pharmaceutically acceptable vehicle that will not be harmful if incontact with a tissue surface. Buffers, if employed, are preferably inthe physiological pH range of 7.2-7.4 but may also be at lower pH suchas provided by acetate buffers or at a higher pH in more alkalinebuffers such as carbonate buffer. For many applications thesuperoxygenated compositions will comprise water and oxygenmicrobubbles.

It may be beneficial in some circumstances to provide agitation to thesuperoxygenated composition while it is being applied to the tissue.This will increase oxygen contact to the tissue surface and may increaseefficiency of uptake. Agitation is inherent in the method of applicationwhen the compositions are part of a whirlpool bath treatment and maycompensate somewhat for some decrease of oxygen in an open atmosphereenvironment and use of temperatures that are intended to provide patientcomfort.

The oxygen supersaturated compositions of the invention may be appliedin a variety of ways depending on the area to be applied, the nature ofthe condition and, for treatment purposes, the health condition of thesubject or patient to be treated. Skin treatments will typically beapplied as solutions that may be incorporated into creams, pastes,powders, ointments, lotions or gels or simply superoxygenatedmicrobubble preparations in nonaqueous or aqueous media. An importantconsideration will be the concentration of microbubbles in thepreparation and its ability to increase subepithelial partial oxygenpressure.

The method of application to the skin may be by soaking, immersion,spraying, rubbing or aerosols. The preparations may be applied todressings that are in contact with the skin, such as plasters and woundcoverings. In other applications, douches or enemas may be used forvaginal or rectal administration. Selection of the method will depend onparticular patient needs, the area of application and type of equipmentavailable for application.

Superoxygenated compositions are another aspect of the invention. Thecomposition comprises an aqueous-based solution of oxygen microbubbleshaving a diameter of from about 0.6 micron to about 100 microns andhaving an oxygen concentration between about 45 ppm and about 220 ppm.Preferred embodiments include superoxygenated compositions where themicrobubble diameter ranges from about 0.6 to about 5 microns andcompositions where the microbubble diameter is about 5 to about 8microns. A highly preferred superoxygenated composition includesmicrobubbles of oxygen in the range of 1-2 microns.

While liquid microbubble superoxygenated compositions will be preferredin most applications, the compositions may be in solid or frozen form.In aqueous based solutions this may be as low as −40° C. but could be aslow as −70° C. in frozen gases such as carbon dioxide or in liquifiedgases such as nitrogen. These low temperatures are not practical forapplications to living tissue; however, long term storage of certaincells or other biological material may benefit from this type ofenvironment. In any event, there are several applications ofsuperoxygenated aqueous solids in providing for example a slow releaseoxygen environment or where ice might be in contact with excised organsbeing transported for transplant purposes.

The compositions and methods disclosed may be combined in an apparatusfor the purpose of providing a tissue oxygenating environment to amammal in need of increased tissue oxygenation. An apparatus may includea container for holding an at least 55 ppm superoxygenated aqueoussolution produced from an oxygen generating machine connected to thecontainer. The apparatus may further include additional features formore efficient and convenient use, such as devices to agitate thesuperoxygenated composition being applied. In a particular embodiment,the device may induce a whirlpool effect. The device may be a sonicatorto provide more effective distribution of microbubbles and which mayhelp to maintain high oxygen levels in the solution. Stirrers, shakers,bubblers and the like may also be used to provide mixing.

The apparatus may also include a temperature controller that may beuseful in controlling the oxygen levels in the superoxygenatedsolutions. An additional effect may be to enhance oxygen uptake throughthe skin of some subjects due to an increase in skin surfacetemperature. For use with patients, one may prefer to adjusttemperatures to between about 37° C. and about 45° C.

In a particular application, the methods and compositions may be used totreat anaerobic infections. Generally this will involve applying any ofthe aforementioned compositions to a skin lesion suspected of harboringanaerobic bacteria. The method should be particularly effective againstthe anaerobic bacteria typically found in gangrenous or ulceratedtissue. Such anaerobic bacteria are also found in wound infections.Patients are likely to benefit from increased tissue oxygen in the woundarea. Burned skin areas are particularly susceptible to infection,particularly where tissue is destroyed or badly damaged as in second andthird degree burns. Burn patients are expected to benefit from suchtreatment that can be used prophylactically as well as therapeutically.Other conditions that will benefit from increased tissue oxidationinclude the soft tissue in the oral cavity, particularly in treating gumdisease that is usually caused by anaerobic bacteria.

For convenience, kits may be used to package various superoxygenatedcompositions prepared in accordance with the invention. An exemplary kitwith appropriate instructions for use in topically increasing tissueoxygenation may contain a sealed permeable flexible container and acontainerized superoxygenated composition in one or more of thevariations described. The kits may additionally include a whirlpoolgenerating device, and/or a thermostat/heating device for adjustingtemperature inside the container.

As discussed, the disclosed methods employ application of asuperoxygenated composition to a surface for a time sufficient toincrease the subepithelial tissue partial oxygen pressure (pO₂) fromabout 30% to about 120% above baseline pO₂ levels. The method isapplicable particularly to humans who suffer from such conditions astissue necrosis, bedsores, ulcers, burns or anaerobic infection.

The present invention addresses several of the problems encountered inattempts to develop therapies and treatments that increase topicalavailability of oxygen to tissues, particularly to the skin. Skinconditions, such as ulcers, bedsores, wounds, burns, and other seriousdermatological problems may be treated by utilizing an aqueous solutioncharged with oxygen microbubbles applied directly to the skin. Animportant application is scar reduction where treatment may be usedsubsequent to scarring or on wounds, burns or surgical incisions toreduce scar formation. The methods are also applicable to increasingoxygen levels in infected surface tissues such as puncture wounds andsoft tissue infections of the oral cavity.

It is well known that many types of skin sores, ulcers, wounds and burnsdo not heal properly because there is a severe depletion of oxygenreaching these affected areas due to degeneration or damage of theassociated blood microcirculation. The human skin is at the terminus ofthe oxygen delivery system and exhibits signs of oxygen loss in avariety of pathological conditions. Degeneration of skin tissue islargely due to oxygen deprivation. Although the skin is exposed to theatmosphere, only a negligible amount of oxygen is actually absorbed.Increasing the level of oxygen absorbed by the skin directly results inincreased healing rates of the skin.

The present invention utilizes a method of tissue superoxygenation thatprovides oxygen to tissue to aid in its healing and revitalization.Oxygen is provided to the tissue through microscopic bubbles and ispresent at a pressure many times that found in blood. The oxygen in themicrobubbles can be transported through the skin when placed in contactwith the skin. Such treatment increases the oxygen level in theinterstitial fluids of the subepithelial and dermal tissues and isimmediately available to the oxygen-depleted cells, thereby inducingmore rapid healing. The disclosed procedures will aid in the preventionof gangrene formation and treatment of sepsis, decrease the need foramputations in diabetic patients, and help to heal bedsores, skinlacerations, burns and wounds. This type of treatment is more convenientto use and is much more affordable than existing methods of treatmentfor these conditions, such as a hyperbaric chamber.

The methods are useful not only in prevention of several skin disordersbut also in cosmetic and pharmaceutical applications. Of particularinterest to many teenagers and even adults are formulations that willbenefit healthy skin while also promoting healing of common acne, a skincondition that may be disfiguring to a certain degree.

Superoxygenated compositions may also benefit victims suffering fromsmoke inhalation and damage from inhaled hot air. In such cases, thedisclosed superoxygenated compositions are administered directly to thelung in order to increase oxygen concentration to the damaged cells.Such treatment may also be used to wash inhaled particulates from thelungs and can be administered in conjunction with antibiotic andanti-inflammatory drug solutions where indicated. The superoxygenatedfluid can be used as a spray or intubated as a soaking solution toprovide more controlled contact with the internal surface of the lung.

In like manner, internal injuries such as bullet wounds may benefit frombeing flushed with the superoxygenated fluids herein disclosed. Thiswill be particularly useful for deep wounds where surgery is notindicated or in field situations where access to the wound is difficult.In such cases, the wound is flushed with the disclosed compositions toinhibit anaerobic infection and to provide supplemental oxygen todamaged tissue.

The superoxygenated compositions are typically aqueous solutions ofoxygen microbubbles with diameters from about 0.1 to about 10 microns,preferably about 1 to about 8 microns and more preferably at least about0.6 to about 8 microns with oxygen concentrations from about 45 ppm toabout 220 ppm. In most applications the solutions will includemicrobubbles with a range of sizes, including less than 0.6 microns upthrough 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 microns and may contain largermicrobubble sizes as microbubbles coalesce, depending on temperature. Ofcourse the oxygen concentration will depend on the temperature of theliquid, typical oxygen concentrations being up to about 220 ppm at 2° C.or about 118 ppm at 34° C. These concentrations may be varied dependingon the condition of the tissue surface to be treated, the type of tissueand the location of the tissue surface.

In special applications considerably higher oxygen concentrations may bedesired; for example, well above 220 ppm. This may be achieved bypreparing solutions of oxygen nanobubbles as small as 20-30 nanometerssuch as those described in association with flowing liquids acrosshydrophobic surfaces (Tyrrell and Attard, 2001). Nanobubbles are thoughtto be flat rather than round and to form closely packed, irregularnetworks that nearly completely cover hydrophobic surfaces. They appearto reform quickly after being distributed and are therefore quitestable. Regardless of how nanobubbles are produced, it is likely thatconcentrations of oxygen significantly higher than 250 ppm may beattained and will be useful in achieving high tissue oxygenation levels.

Oxygen microbubbles may be prepared in water or in a pharmaceuticallyacceptable vehicle. Physiological saline, various buffers, or compoundsthat increase wetting and porosity are examples of compositionvariations. In some cases, one may wish to add antibiotics,anti-inflammatory compounds or other drugs to the compositions in orderto expedite healing or more effectively treat certain bacterialinfections.

In certain applications, it may be desirable to administersuperoxygenated compositions in the form of creams, lotions, gels orsolids. Such formulations are well recognized and accessible to thoseskilled in the art. The superoxygenated compositions may also bemaintained in a frozen state, for example for storage, or for use intreatments where ice can be conveniently applied to a tissue surface sothat higher levels of oxygen can be consistently maintained. In aparticularly important application, frozen or chilled superoxygenatedcompositions may be used for storage and transport of organs intendedfor transplantation. This may avoid or ameliorate anoxic conditionsarising from severance of the organs from the normal blood supply.Frozen or chilled compositions will be especially beneficial for suchtissues, both because enzymatic processes are retarded at the lower thetemperature, and because at lower temperatures, higher levels of oxygencan be incorporated into the oxygenated compositions so that degradationis inhibited.

The superoxygenated compositions may be administered in several wayssuch as through tubes connected to flexible bags containingsuperoxygenated solution or in some applications by immersion of tissuein a bath containing the oxygenated solution. For dental applications intreating gum disease, administration by a device similar to a water picis an effective method for topically administering suitablesuperoxygenated solutions. Certain applications benefit from mixing oragitating procedures so that fresh solution constantly bathes thetissue; for example, lavage procedures or whirlpool baths in which anaffected limb is immersed.

In certain embodiments, an apparatus for providing a tissue oxygenatingenvironment to a mammal in need of increased tissue oxygenation is alsowithin the scope of the invention. Such an apparatus incorporates amachine for generating oxygen microbubbles that may be as simple as anoxygen cylinder connected to a pressurized vessel at pressures in therange of 90-110 psi and introducing oxygen gas into the vessel thatholds a liquid such as water or other suitable water-based fluid. Anoxygenator may also be used, generating about 50 psi. A tube or otherexit from the vessel provides the oxygenated solution to the targettissue. Oxygen levels in the solution may be increased by agitating orsonicating the vessel. Ultrasonic equipment external to the flow intakeand adaptations to control diffusion patterns in a vessel or a bath mayalso be employed.

It will be appreciated also that solution temperature will affect totaloxygen concentration so that in alternative embodiments, the apparatusmay incorporate any of a number of well known devices for controllingtemperature such as thermostatted baths. Thus where applications arewhole limb or body applications in open air as in a whirlpool bath,oxygen concentrations will not usually exceed about 55 ppm. Fortreatment of an internal epithelial lining, as in oral mucosalinfections, cooler temperatures and correspondingly higher oxygenconcentrations will be tolerable. Oxygen concentrations will varydepending on the method of application whether by soaking, immersion,spraying, rubbing or aerosols; however, in any event, the compositionscontacting the affected tissue will have a significantly increasedoxygen concentration in the range of at least about 45 ppm.

While most applications will utilize aqueous solutions, the inventors donot wish to be unduly limited since high oxygen concentrations may beachieved in nonaqueous or aqueous/organic solvents. Such solvents shouldbe non-toxic and pharmacologically acceptable for human use.Perfluorocarbons are a particular example of non-aqueous solvents thatmight be useful. Other solvents include those that are water-misciblesuch as alcohols and glycols. In certain applications it may beconvenient to use gel formulations such as hydrophilic gels formulatedfrom alginates or carrageenans.

Other embodiments include kits that conveniently provide some form ofthe apparatus described above and will be useful for topicallyincreasing tissue oxygenation. Exemplary kits may include a sealedpermeable flexible container containing a superoxygenated compositionand instructions for applying the composition to the tissue surface orskin requiring increased oxygenation. Optional kit components include athermostat/heating device for adjusting temperature inside the containerand an oxygen supply connectable with a pressurized vessel for mixing,agitating or sonicating an oxygenated fluid.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thefollowing drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1 Illustrates oxygen release to cells

FIG. 2A Illustrates a cross-sectional view of normal skin. Arrows shownormal direction of diffusion of oxygen from capillaries into dermis andoverlying epidermis.

FIG. 2B Illustrates a cross-sectional view of abnormal skin. Thesuperoxygenated composition of the invention is applied to the surfaceof the skin; arrows indicate direction of movement of oxygen through theepidermis and into the underlying subcutaneous tissues.

FIG. 3 Shows the size distribution of microbubbles in thesuperoxygenated composition.

FIG. 4 Shows measurements of subcutaneous pO₂ levels in pig skin,indicating rapid diffusion of oxygen through the skin following topicalapplication of oxygen microbubbles. Topical application of controlsolution had no effect.

FIG. 5 Shows comparison of pO₂ increase and skin temperature, indicatingincrease in pO₂ following topical application of oxygen microbubblesalone, with comparable skin temperatures following application of testor control solution.

FIG. 6 Shows percent increases from baseline subcutaneous pO₂ in humansubjects during control and oxygenation periods during immersion oftissue in a whirlpool bath.

FIG. 7 Shows subcutaneous pO₂ increase over time in subject HY01 duringimmersion of leg in a whirlpool bath.

FIG. 8 Shows subcutaneous pO₂ increase over time in a subject HY07during immersion of leg in a whirlpool bath.

4.0 DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

There is a need for improved treatments for skin disorders such asgangrene, skin ulcers, bedsores, burns, and other serious dermatologicalproblems. The present invention utilizes the application of a highlyoxygenated product in the treatment of several skin disorders. Thedisclosed highly oxygenated products will be useful in treatment of skindiseases related to degeneration of skin tissue due to oxygendeprivation, such as ulcers, burns and skin wounds.

4.1 Oxygen Release to Cells

As shown in FIG. 1, oxygen is transported from the air into the body.Air, which contains approximately 20% oxygen, passes upon inhalationinto the bronchial tubes and ultimately into the alveoli of the lungs.In the alveoli, oxygen diffuses across very thin capillary walls toenter the bloodstream, where it combines with hemoglobin in the redblood cells to form oxyhemoglobin. As the blood circulates through thebody, oxygen is released from the oxyhemoglobin and diffuses into thetissues and cells of the body, including the skin.

Gases are usually measured in terms of pressure. Air is a mixed gas andis measured in terms of absolute and partial pressure. For example, atsea level air has an atmospheric pressure of 760 mm of mercury (Hg),meaning that it will support a column of mercury 760 mm high in a tube 1mm in diameter. Oxygen makes up 20% of the gases in air; thus thepartial pressure of oxygen (pO₂) is 20% of 760, or 152 mm Hg. At higheraltitudes, the pO₂ of air is decreased. In the lungs, the partialpressure of oxygen is 100 mm Hg.

The diffusion of oxygen into cells and tissues depends on the partialpressure of oxygen, the solubility of oxygen in the body fluids and onthe health of the tissue. Oxygen does not penetrate the skin atatmospheric pressure, but only interacts with the outer surface. Thusunder normal conditions, the skin is nourished not from oxygen in theair, but from O₂ that diffuses from beneath into the deep, living layersof the epidermis and the underlying dermis from capillaries in thedermis (FIG. 2A). Compromise to the blood supply of the skin throughdamage or disease thus severely affects the ability of the damaged skinto obtain an adequate oxygen supply.

Hyperbaric oxygen therapy is a systemic treatment to increase tissueoxygenation involving administration of oxygen at pressures higher thanatmospheric pressure. This requires the use of a special chamber tocontain the high pressure (usually between 2 and 3 times atmospheric)which is needed to force extra oxygen to dissolve in the plasma, whichin turn forces it into the tissues. To date, the majority of skinconditions resulting from lack of oxygen are treated with systemichyperbaric methods and nonoxygenated topical applications. For example,the hyperbaric oxygen chamber has been established as the primarytherapy in the treatment of medical disorders such as ClostridialMyonecrosis (Gas Gangrene). On average, treatments usually last from 1to 2 hours at full pressure, which may be problematic because extendedexposure to hyperbaric treatment at these pressures produces high risksof toxicity.

Hyperbaric oxygen therapy is also used to treat bedsores. Skin has arich blood supply that delivers oxygen to all its layers. If that bloodsupply is cut off for more than two or three hours, the skin begins todie, beginning at its outer layer, the epidermis. A common cause ofreduced blood flow to the skin is pressure. Normal movement shiftspressure and enables the continuous movement of the blood supply. Once aperson is limited in movement or bedridden they are at a high risk fordeveloping bedsores. Bedsores can further develop into decubitus ulcers.These ulcers can open skin to the bone, causing a great deal of pain andcan result in a life-threatening situation.

In some cases, “topical hyperbaric” treatment for bedsores, involvingexposure of an isolated portion of the body to oxygen gas, is claimed tobe effective during the early stages of infection There is concern withthis method that there is lack of penetration of the topically appliedoxygen, largely due to the difference in the pressure under the surfaceof skin and the atmosphere (FDA Advisory Meeting, Nov. 17, 1998). Alsooxygen delivery topically causes a burning effect on the surface of askin after continuous application to skin.

In the present invention, a method of tissue oxygenation usingsuperoxygenating compounds has been developed to treat dermatologicalproblems by inducing more rapid healing. The tissue is provided withoxygen by a method utilizing topical application of highly oxygenatedwater or other fluid incorporating microscopic oxygen bubbles. Whenapplied for example to skin, the oxygen is transported inward from thesurface through the deeper layers of the skin (FIG. 2B), therebyproviding oxygen to the cells of the epidermis and underlying dermis.The highly oxygenated solutions will increase the level of oxygen in thesubcutaneous and subepithelial tissues and promote healing by providingoxygen to oxygen-depleted cells.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Materials and Methods

Superoxygenated solutions were prepared according to processes forenriching a liquid with oxygen by introducing the liquid into an oxygenenriching vessel similar to the disclosure of U.S. Pat. No. 5,006,352,hereby incorporated by reference. Briefly, the oxygenation process iscarried out in an oxygen enriching apparatus as disclosed in U.S. Pat.Nos. 5,766,490 and 5,814,222, herein incorporated by reference in theirentirety.

The process utilized by the inventors introduces a liquid into a closedspace or pressurized vessel, mixes the liquid with oxygen in a turbulentmixer and recovers an oxygen-enriched liquid with an oxygen content ofat least 40 mg/l oxygen. A superoxygenated fluid having an oxygenconcentration of 180-217 ppm was prepared using distilled water. Thissolution contained oxygen microbubbles with diameters averaging about 1micron and usually in the range of 0.6-8 microns, as measured using aflow impedence device. For measurement of bubble diameters oxygenatedwater samples were dispersed by mixing equal amounts of each with IsotonII in 20 ml cuvettes. Analyses were performed with a 30 μm aperture tubeusing Time-mode for 30 seconds. Table 1 summarizes results showing thatthe mean size of the oxygen bubbles was in the range of 1 μm. Attemptsto measure particle size using a laser diffraction instrument wereunsuccessful.

TABLE 1 Particle Size VOLUME MEAN SIZE NUMBER MEAN SIZE SAMPLE (μm) (μm)119-155 6.29 1.30 107-123 2.21 1.07 180-216 9.31 1.11

Distribution of particle size in a typical sample is represented in FIG.3. The graph shows particle distribution in terms of solution volume,indicating that in the sample 90% of the particles were 1-2 μm indiameter.

5.1 Example 1 Increase in Subcutaneous Oxygen in Porcine Skin

This example demonstrates that a solution containing superoxygenatedmicrobubbles, applied topically to the skin of a pig, increases thelevel of oxygen in the underlying subcutaneous tissue.

The skin was cleaned with alcohol, hydrogen peroxide and then water.Arterial blood gas monitoring devices (“sensors”) capable ofsimultaneous measurement of partial pressure of carbon dioxide (pCO₂),temperature and pH were inserted 1-4 mm beneath the skin surface, on theleft and right sides of the body. Containers for test solutions, such asflexible bags with tubes at one end, were affixed with adhesive to theskin surrounding the sensor. The containers provided a means ofimmersing the skin under a column of test or control solution during thetest. Prior to filling the containers, controls established that thechoice of adhesive (Fixodent® or Stromahesive paste) had no effect onthe baseline pO₂ reading.

The sensors were allowed to equilibrate for 30 minutes and both werestable before the interventions were begun. Control and test solutionswere heated to 34° C., and equal volumes were added to the container toensure equal pressure on the skin site during measurement. Controlsolution was distilled water (approx. 7-9 ppm O₂) and the test solutionwas superoxygenated water having an O₂ content of 180-217 ppm.

As shown in FIG. 4, at the start of testing, baseline pO₂ was 55 mm Hgin the sensor positioned at the test site on the left side. The controlsolution was placed in the container for 6 min, during which interval noincrease in pO₂ was seen (FIG. 4). The control solution was removed andtest solution was placed in the container at t1 and again four minuteslater (second dotted line). Addition of the test solution resulted in anincrease in pO₂ from 54 to 117 mm Hg. This tissue level was 23% higherthan the arterial pO₂ value of 95 mm Hg. (FIG. 4). As soon as the testsolution was removed by suction, the pO₂ returned to baseline.

The cycle was then repeated. Control solution was placed in thecontainer thirty minutes later (12:30) and more was added to keep theskin surface covered. No increase in pO₂ was observed. Subsequently, thecontrol solution was removed and replaced with several additions of testsolution (dotted lines). With additions of test solution, there werepeaks in pO₂ levels (FIG. 4).

Sensor response is known to be affected by temperature. It was importantto determine the relationship between the pO₂ increases and the skintemperature at the test site. As seen in FIG. 5, both the control andtest solutions altered the skin temperature by 1-2° C. Because the pO₂increased only with the superoxygenated test solution, it was concludedthat the increases in pO₂ were not due to changes in temperature butwere actually due to the application of the test solution to the surfaceof the skin.

A second test using the sensor implanted on the right side of the animalrevealed the importance of the placement of the electrode. Differenceswere not observed between the pO₂ levels recorded with control and testsolutions. However, the baseline pO₂ at the site was 75 mm Hg, muchcloser to the blood pO₂ level of 90 mm Hg, (as compared with 55 mm Hg onthe left side site). This may have been due to the sensor being in closeproximity to a capillary. Gas exchange from the blood may have causedthe differential between the flowing blood and the test solution to betoo small to observe. Alternatively, the sensor may have been placed toodeep to detect diffusion of O₂ through the skin.

5.2 Example 2 Increase in Subcutaneous Oxygenation in Human Subjects

Results from procedures with human subjects demonstrated that oxygen insuperoxygenated solutions prepared as described can be delivered tosubcutaneous tissue through healthy human skin to increase subcutaneouspO₂ above baseline levels.

After receiving informed consent from ten human subjects, baseline bloodpressure and heart rate were measured. A pulse oximeter probe was placedon the subject's finger for continuous monitoring of heart rate andoxygen saturation throughout the study. The skin over the outside of theleft calf was disinfected with betadine and the betadine cleaned fromthe surface of the skin. A catheter (22 Ga, 1⅜″ catheter, Product No.04122, Arrow) was surgically inserted in under the surface of the skinand then out, so the tip of the needle and catheter were exposed andalmost the entire length of the catheter was in the subcutaneous space.The catheter was placed as close to the surface of the skin as possiblewithout driving the needle through the skin prematurely, in order toplace the catheter in the dermis or at the border of the dermis andsubcutaneous tissue.

After the catheter was placed, the needle was removed and the tip of thesensor was lined up with the tip of the catheter. The sensor wasadvanced through the catheter using the sensor advancement mechanismuntil it was visible from the other end of the catheter. The catheterwas removed and the sensor was drawn back until the tip of the sensorjust disappeared beneath the skin. The sensing element was 2.5 cm longand was completely contained beneath the skin. The holes made by thecatheter were covered by a water resistant dressing (Duoderm® thin,Product number NDC 0003-1879-55, ConvaTec) and the sensor housing wastaped in place to keep the weight of the housing and cable from pullingthe sensor out.

The skin around the sensor was prepped with a barrier wipe (Allkare®,ConvaTec). Stoma paste (Stromahesive, Product number NDC 0003-1839-10ConvaTec) was placed on the skin where the flexible bag (ActiveLife®,Product number NDC 0003-0254-33, ConvaTech), with a 2.5″ hole, was to beplaced. The bag was then placed on the applied paste. Any gaps betweenthe bag and the skin, particularly around the sensor housing, werefilled with stoma paste. The bag was also sealed to the skin with Hytapearound the sensor housing.

Tests were performed in two phases, the first phase involving testing ofresponses to control and oxygenated solutions applied over the sensorsites by means of attached bags, as in Example 1. The second phase,carried out after the first on the same patients, involved removal ofthe bag while maintaining the sensor in place, to enable subsequenttesting of responses when the legs of the subjects were immersed in testsolutions in a whirlpool bath. The whirlpool bath was a standardstainless steel bath approximately 30″ long×18″ wide×30″ high of thesort typically used for physical therapy and in other clinical settings.

In phase one, two different concentrations of oxygenated water and acontrol solution in the bag. The oxygenated water for this test wasprepared as previously described. The sensor was allowed to equilibratefor 30 minutes after insertion. The sensor simultaneously measured andrecorded temperature, partial pressure of oxygen (pO₂), carbon dioxide(pCO₂) and pH. Data were collected every 10 seconds on a laptop computerthroughout the entire study. Individual bottles containing thesesolutions were heated to 32° C. (except for a few subjects; see Table2). The order of these solutions was randomized. 500 ml of the selectedsolution was placed in the flexible bag, which was then closed with theclip. The solution remained in contact with the skin for 15 minutes. A15 minute stabilization period was maintained between each solution. Notall subjects received 3 solutions (see Table 2). At the completion ofthis phase the bag was cut from the adhesive frame to expose theskin-covered sensor to air and eventually to water in the whirlpool.

For the second (whirlpool) phase, the procedure for producing theoxygenated solutions was modified as follows. Oxygen is known todissipate rapidly from solutions in open vessels. In order tocontinuously maintain elevated oxygen levels in the solutions as thesolution circulated in the open bath, the outflow from the bath wasconnected to the inlet of the oxygenation machine, which allowed forrecirculation at a rate of 35 gal/min, and return of all of the water tothe bath every 2 minutes. Both control and oxygenated solutions weresubjected to pressures of 90 psi within the machine, and continuouslycirculated throughout the tests. Oxygenation of the circulating waterwas achieved rapidly after activating the oxygen input in the machine,and the level of O₂ was monitored throughout the test by a dissolvedoxygen meter in the bath. The presence of the oxygen in the water wasalso detected visibly by the change in its appearance to a milky whitesolution.

The tape covering the sensor was removed and the bag adhesive pulledback part way. The sensor was withdrawn from the skin using the sensorretraction mechanism. The subject's blood pressure and heart rate weremeasured at the end of the study. In three of the subjects the thicknessof the dermis and depth of the sensors were measured using a 20 MHzultrasound system from GWB International.

At the beginning of this period the sensors had been in the tissue atleast 1 hour. When the leg was inserted into the heated whirlpool thetemperature quickly (within <1 min) rose to the level of the bath. Thebaseline readings for temperature, pO₂, pH and pCO₂ were recorded justat the time the temperature stabilized to the bath level. Controlreadings and subcutaneous oxygenation readings were taken at the end ofthe period for each subject. Statistical analysis was performed on thedata collected during the whirlpool phase. Changes in temperature andsubcutaneous pO₂ between baseline, control and oxygenation periods werecompared with a repeated-measures Anova test, followed by a Tukey HSDtest to elucidate differences between the time periods.

Five male and five female subjects were tested in this study. Table 2details the specific characteristics of each of the subjects and theprotocol. All subjects would be considered overweight (BMI 25). Subjectsreceived slightly different treatment before the whirlpool study.However every subject had at least 1 hour sensor stabilization timebefore the whirlpool study began and every subject spent approximately30 minutes in the oxygenated water. All statistical analysis was limitedto the time period in the whirlpool.

TABLE 2 Subject characteristics and specific protocols test soln 1 testsoln 2 test soln 3 total time before whirlpool cntrl whirlpool O2 (m)Temp Subject Gender Age BMI (15 m) (15 m) (15 m) whirlpool (m) (m) ~4ppm ~55 ppm (C.) HY01 F 41 35.1  4 ppm 4 ppm 112 ppm 109 11 24 32 HY02 F42 31.1 120 ppm 115 ppm   4 ppm 105 16 34 32 HY03 M 43 26.5 101 ppm 4ppm 90 17 30 32 HY04 M 46 25.8 131 ppm 118 ppm   4 ppm 121 16 33 32 HY05F 41 34.0 116 ppm 4 ppm 120 ppm 127 15 36 32 HY06 M 53 27.0 134 ppm 4ppm 92 30 30 32 HY07 F 54 30.1 111 ppm 4 ppm 92 30 33 34 HY08 M 41 31.9131 ppm 60 30 34 35 HY09 F 44 33.6 150 ppm 61 30 26 37 HY10 M 40 33.0130 ppm 60 30 33 34

Table 3 details a qualitative assessment of the sensor depth, withquantitative measurements for the 3 subjects with studied withultrasound. Based on the ultrasound measurements, if the sensor could befelt as a bulge through the skin, it is likely that the sensor was atthe interface between the dermis and underlying subcutaneous tissue. Inone subject (HY03), the sensor came out because it failed to be lockedin place. This subject had the sensor re-inserted in the same region oftissue. Another subject (HY09) had bleeding when the sensor was insertedand a blue line of blood was observed under the skin, along the sensor,suggesting that this sensor may have been sitting in blood. In a thirdsubject (HY10) there was bleeding when the sensor was removed,suggesting that there may have been blood in the channel for thissubject as well.

TABLE 3 Sensor depth assessment and notes on specific subjects SubjectSensor Depth Notes HY01 could see bulge below skin first test solution39° C. HY02 Could not see below skin sensor deep HY03 could see bulgebelow skin reinserted sensor HY04 could feel sensor under skin HY05Could not feel, 2-3 mm deep Ultrasound before whirlpool HY06 could feelsensor under skin, Ultrasound before and after ~1.5 mm deep sensorplaced HY07 could feel sensor under skin, Ultrasound after sensor placed~1.5 mm deep HY08 could feel sensor under skin HY09 could feel sensorunder skin bleeding when put in catheter, blue line along sensor HY10could feel sensor under skin some bleeding observed on removal

For the whirlpool phase of the testing, the bath was heated to 32° C.(except for a few subjects, see Table 4). For the first 5 subjects, theleg was immersed in the bath in control solution (distilled water at 4ppm O₂) for 15 minutes. For the last 5 subjects, the control phaselasted 30 minutes. After the control period, the oxygenation machine wasturned on to oxygenate the water. Full oxygenation (˜55 ppm) was reachedin 3-4 minutes and the leg was immersed for 30 minutes in oxygenatedwater. The subject's leg was then removed from the bath and followed for15 minutes.

Plots of temperature and subcutaneous pO₂ throughout the entire protocolfor selected subjects are shown in FIG. 6 and FIG. 7.

TABLE 4 Temperature and subcutaneous pO₂, pH, and pCO₂ for the whirlpoolprotocol PO₂ PO₂ PO₂ temp temp temp PCO₂ PCO₂ PCO₂ pH pH pH Subjectstart cntrl wO2 start cntrl wO2 start cntrl wO2 start cntrl wO2 HY01 3837 85 33 33.6 34.6 33.3 34.9 38.3 7.44 7.41 7.38 HY02 22 18 31 31.4 33.135.7 32.1 34.3 38.7 7.47 7.46 7.42 HY03 28 20 18 32.7 33 35.6 29.6 30.535.7 7.46 7.45 7.40 HY04 21 20 27 32 32.1 33.4 41.5 42.7 46.5 7.41 7.407.38 HY05 11 10 19 31.3 32.2 33.1 36.1 36.1 39.0 7.45 7.44 7.41 HY06 4040 49 32.3 32.5 34.1 34.3 34.2 36.2 7.46 7.45 7.43 HY07 22 29 70 32.734.1 34.3 40.3 40.0 40.4 7.43 7.42 7.39 HY08 28 24 32 35.1 35.2 35.441.4 44.1 46.8 7.40 7.37 7.35 HY09 53 54 60 37 36.7 36.5 37.5 39.7 41.77.41 7.38 7.30 HY10 42 42 47 34.1 34.2 34.4 33.8 34.4 35.7 7.46 7.45 7Mean 31 29 44 33.2 33.7 34.7 36.0 37.1 39.9 7.44 7.42 7.39 Std dev 13 1422 1.8 1.4 1.1 4.1 4.3 4.1 0.03 0.03 0.04

A summary of the whirlpool data for all subjects is shown in Table 4.The mean pO₂ for the 10 subjects started at 31+/−13 mm Hg, but was notsignificantly different at the end of the control period (29+/−14 mmHg). After immersion in the oxygenated water, the subcutaneous pO₂increased significantly to 44+/−22 mm Hg (p≦0.026 compared to baselineand p≦0.016 compared to the end of control). The percent increase (ordecrease) in subcutaneous pO₂ during the control and oxygenation periodsfor each of the subjects is shown graphically in FIG. 8.

From FIG. 8 it can be seen that the percent increase in pO₂ variedconsiderably among the subjects, with 6 of the 10 showing increases insubcutaneous pO₂ of at least 30%. Significantly greater increases (141%,130%, 90%, 70%) were observed for subjects HY07, HY01, HY05 and HY02,respectively. From Table 3 it can be seen that there is an increase intissue temperature during both the control and oxygenation phases of thestudy. The temperature at the end of the oxygenation phase issignificantly different from baseline (p≦0.0006) and from the end of thecontrol period (p≦0.015). These temperature changes are accompanied byan increase in tissue pCO₂ and a decrease in tissue pH. Table 5 showsthe average percent increase in baseline values for each of theparameters measured. In this analysis pH was converted to hydrogen ionconcentration [H⁺].

TABLE 5 Average percent increase from baseline at the end of the controland oxygen periods Average percent increase from baseline Control WithO2 Temperature 2 5 P_(SC)O₂ −4 44 P_(SC)CO₂ 3 11 [H+] 4 12

The data show that there is a significant increase in subcutaneousoxygen tension when the subject's leg is immersed in the oxygenatedwater, when compared to either baseline or control values in which thewhirlpool contained regularly oxygenated (˜4 ppm) water. Coincident withthe subcutaneous pO₂ increase is an increase in temperature. Theincrease in temperature does not appear to affect the sensor response(which corrects for changes in temperature) but does have a number ofphysical and physiologic effects. As temperature increases, thesolubility of oxygen in the blood decreases, increasing its release tothe tissue. Also, temperature increases result in enhanced dissociationof oxygen from oxyhemoglobin. Lastly, increases in temperature result invasodilation, which should bring more oxygen to the tissue.Nevertheless, for subjects HY01, HY05, HY07, HY08 and HY10, theincreases in pO₂ seemed to be due to sources beyond those caused by atemperature rise, where for all subjects except HY01 the temperaturerise was less than 1° C. It is possible however that the observed pO₂increases may have been due to temperature in subjects HY02, HY04 andHY06 where the squared correlation coefficient (R²) between pO₂ andtemperature is above 0.8° C.

The levels of pCO₂ and [H⁺] increase during the period when thecirculating water is being oxygenated. Both increase the same amount,which is not surprising given their dependence through the bicarbonateequilibrium. The source for these changes was not determined.

One subject (HY03) showed a decrease in pO₂ as a result of oxygenation.In this subject the sensor had to be reintroduced a second time. Injuryto the tissue may have resulted in an impaired response to oxygen. Asecond subject (HY09) had obvious bleeding when the sensor was insertedand the sensor may have been insulated from the interstitial fluid byblood, dampening the response to the added oxygen. Some, though less,blood was observed when withdrawing the sensor from subject HY10 and mayexplain a small response from this subject as well. When the sensor isinserted in tissue, ample time must be allowed for temporary tissueinjury to subside. Initially 30 minutes was believed to be acceptable,but continued decline after that period indicated that all injury wasnot resolved. Taking baseline readings at the beginning of the whirlpoolperiod allowed at least 60 min stabilization for each subject. This timeis consistent with studies conducted in Sweden using the Paratrend®sensor directly in subcutaneous tissue of swine (Mellstrom et al.,1999). The baseline values in those studies were not that different fromthose measured in this study: pO₂: 58+/−16; pCO₂: 42+/−5, and pH:7.46+/−0.06. In a study of surgical patients where a polarographicoxygen electrode was used, baseline subcutaneous pO₂ was found to be43+/−10 (Hopf et al., 1997). These reported values may be slightlydifferent than values in these experiments because those sensors wereprobably placed deeper than the present ones, which were likely near theedge of the dermis.

Results showed that in at least half of the subjects there was asignificant increase in tissue pO₂ related to the introduction of oxygenmicrobubbles in the whirlpool bath. The whirlpool was more effectivethan still oxygenated water (phase 1 of the test) in producing anincrease in subcutaneous pO₂, despite the higher concentration of oxygenin the water used in the flexible bags (101-150 ppm O₂ vs. 55 ppm in thewhirlpool). In the two subjects where there was a very good response inthe whirlpool (HY01 and HY07) there was also a measurable response tooxygenated water in the bags. These were the only two subjects wherethere was a response during the bag portion of the study. These resultsindicate that individual subjects may differ in the rate of diffusion ofoxygen through their skin to the sensor.

Sensor depth was controlled between 1 and 3 mm beneath the surface ofthe skin. However in the three subjects examined with the ultrasound,the structure of the skin and the thickness of the epidermis were quitevariable. If oxygen is diffusion limited, it may only get to a certaindepth. Different subjects may differ in their ability to hydrate or inthe permeability of their skin to gas. Nevertheless, the results of thestudy showed that the high partial pressure of oxygen in the oxygenatedwater permitted a high enough concentration of oxygen outside the skinto facilitate diffusion of oxygen through the skin of the majority ofthe healthy subjects. Furthermore, permeability of the skin would not bea problem for the treatment of open wounds.

5.3 Example 3 Application of Method of Tissue Superoxygenation to WoundHealing

Preliminary studies will be conducted in diabetic patients and comparedto those performed in animal and normal human testing to determine theeffect of superoxygenated microbubbles on the rate of healing whenadministered to the non-healing wounds of diabetic patients. Patientswill be maintained under tightly-controlled environmental conditions.Additionally, the wound area will be analyzed and anaerobic bacteriaidentified according to studies performed at the Institute of MolecularBiology and Medicine at University of Scranton. According to that study,approximately 10-20% of diabetic foot wounds fail initial antibiotictreatment. It is generally believed that several bacterial species maybe present in these types of wounds. Because some of these organismscannot be easily cultured, proper identification is problematic andthus, appropriate treatment modalities cannot be applied. The reportexamined the bacterial flora present in a chronic diabetic foot woundthat failed antibiotic treatment. A tissue sample was collected from thebase of the wound and used for standard microbiological culturing. DNAfrom the sample was used to amplify bacterial 16 S rDNA gene sequencesand prepare a library, the clones of which were sequenced. Theculture-based method identified a single anaerobic species, Bacteroidesfragilis, whereas the method employing rDNA sequencing identified B.fragilis as a dominant organism and Pseudomonas (Janthinobacterium)mephitica as a minor component. The results indicated that the rDNAsequencing approach can be an important tool in the identification ofbacteria from wound (Redkar et al., 2000).

Experiments will be conducted in controlled randomized fashion byadministering the compositions to the wound area in varyingconcentrations and forms with subsequent analysis of the bacteriapresent in untreated wounds and those treated with superoxygenatedwater.

5.4 Example 4 Tissue Superoxygenation in Treatment of Leg Ulcers

A clinical study was undertaken to investigate and compare specificallythe aerobic and anaerobic microbiology of infected and noninfected legulcers. Leg ulcers, defined as infected on the basis of clinical signs,were swab sampled and tested for aerobic and anaerobic microorganismsusing stringent isolation and identification techniques (Bowler et al.,1999).

In this study, 220 isolates were cultured from 44 infected leg ulcers,and 110 isolates were from 30 non-infected leg ulcers. Statisticalanalysis indicated a significantly greater mean number of anaerobicbacteria per infected ulcer (particularly Peptostreptococcus spp. andPrevotella spp.) in comparison with the noninfected ulcer group (2.5 vs.1.3, respectively) (P<0.05). Also, anaerobes represented 49% of thetotal microbial composition in infected leg ulcers compared with 36% innon-infected leg ulcers (Bowler et al., 1999).

Based on the results of these studies, superoxygenated microbubblecompositions will be used to test the effect of the oxygenationtreatment on the distribution of aerobic and anaerobic microflora whichexist in leg ulcers. An indication of the effectiveness of thecomposition in combating leg ulcers and other wound infections will bedetermined by noting the relative changes in distribution of theanaerobes and aerobes.

5.5 Example 5 Superoxygenated Ice

The following example demonstrates the ability of highly oxygenated iceto hold high levels of oxygen and release oxygen at high levels. Resultsindicated that ice can be made with highly oxygenated water and thatboth the ice and melt fluid contain high concentrations of oxygencompared with tap water and ice as controls.

Two bottles of super-oxygenated (SO) fluid were stored at −15° C. Athird bottle was chilled at 8° C. Control samples were ice from tapwater and tap water chilled to 8° C. The superoxygenated fluids werestored for approximately 6 months in tightly capped bottles. Oxygenlevels ranged from 107 to 123 ppm at the time of storage. The bottleswere removed from storage and oxygen levels measured with a modifiedhigh range Oxygard Handy Mk II meter with a standard unit of measurementof parts per million. The meter measured the oxygen at the surface ofthe ice where the ice initially melted ((Ice reading). The melt waterwas also measured (melt reading). The same measurements were made forthe control tap water and tap water ice. Samples at 8° C. were pouredinto an open container and oxygen levels measured directly. Results areshown in Table 6.

TABLE 6 Oxygen Levels in superoxygenated and tap water ice Sample Frozenmelt Stored at 0° C. Superoxygenated water 82 ppm 56 ppm — −15° C.Superoxygenated water 0° C. — — 103 ppm Tap water −15° C.  7 ppm  7 ppm— Tap water 0° C. — —  4 ppm

6.0 REFERENCES

The following literature citations as well as those cited areincorporated in pertinent part by reference herein for the reasons citedin the above text:

-   Bowler, Philip G.; Davies, Barry J., The microbiology of infected    and noninfected leg ulcers, International Journal of Dermatology,    38(8): 573-578, 2000.-   Elden, Harry R.; Kalli, Ted, Hydrogen Peroxide Emulsions, DCI    Magazine, 157(vc): 38, 40, 42, 44, 47, 1995.-   FDA Medical Devices Advisory Committee Meeting of: General and    Plastic Surgery Devices Panel Closed Session, Nov. 17, 1998.-   Hopf H W, Hunt T K, West J M, et al. Wound tissue oxygen tension    predicts the risk of wound infection in surgical patients.    Physiology of wound healing. Arch Surg 132:997-1004, 1997.-   Hunt T, Rabkin J, Jensen J A, et al. Tissue oximetry: an interim    report. World J Surg 11: 126-132, 1987.-   Jonsson K, Jensen J A, Goodson W H, et al. Tissue oxygenation,    anemia, and perfusion in relation to wound healing in surgical    patients. Ann Surg 214:605-613, 1991.-   Ladin, U.S. Pat. No. 5,792,090, 1998.-   Loori, U.S. Pat. No. 5,154,697, 1992.-   Loori, U.S. Pat. No. 5,801,291, 1989.-   Mellstrom A, Hartmann M, Jedlinska B, et al. Effect of hyperoxia and    hypoxia on subcutaneous tissue gases and pH. Euro Surg Res    31:333-339, 1999.-   Moschella and Hurley. Chapter 4: Permeability in Dermatology. W.B.    Saunders; 1992-   Quay, U.S. Pat. No. 5,573,751, 1996.-   Redkar R; Kalns J; Butler W: Krock L; McCleskey F; Salmen A;    Piepmeier E Jr; Del Vecchio V, Identification of bacteria from a    non-healing diabetic foot wound by 16 S rDNA sequencing, Molecular    and Cellular Probes, 14(3): 163-169, 2000.-   Scherson et al., U.S. Pat. No. 5,855,570, 1999.-   Spears, et al., U.S. Pat. No. 6,248,087-   Taylor et al., U.S. Pat. No. 5,766,490, 1998.-   Tegner E and A. Bjomberg, Hydrogen Peroxide Cream for the Prevention    of White Pressure Areas in UVA Sunbeds, Acta Derm, Venerol.    (Stockh), 70:75, 1990.-   Trammell, U.S. Pat. No. 5,029,589, 1991.-   Van Liew et al., U.S. Pat. No. 5,869,538, 1999.-   Whitney J. D., Physiologic Effects of Tissue Oxygenation on Wound    Healing, Heart and Lung 18: 466-474, 1989.-   Zelenak et al., U.S. Pat. No. 5,814,222, 1998.-   Zelenak nee Zoltai et al., U.S. Pat. No. 5,006,352, 1991.    All of the methods and compositions disclosed and claimed herein can    be made and executed without undue experimentation in light of the    present disclosure. While the compositions and methods of this    invention have been described in terms of preferred embodiments, it    will be apparent to those of skill in the art that variations may be    applied to the methods and compositions, in the steps or in the    sequence of steps of the method described herein and in the    modification of the apparatus connected with the methods and    compositions without departing from the concept, spirit and scope of    the invention. More specifically, it will be apparent that certain    agents which are both chemically and physiologically related may be    added to, combined with or substituted for the agents described    herein while the same or similar results would be achieved. All such    similar substitutes and modifications apparent to those skilled in    the art are deemed to be within the spirit, scope and concept of the    invention as defined by the appended claims. Accordingly, the    exclusive rights sought to be patented are as described in the    claims below.

1. A method of increasing tissue oxygenation in mammals, comprising applying a superoxygenated composition of oxygen microbubbles consisting essentially of oxygen in a pharmaceutically acceptable vehicle directly to a tissue surface selected from the group consisting of skin and mucous membranes for a time sufficient to increase the partial oxygen pressure at least about 2 mm beneath the tissue surface from about 30% to about 120% above baseline pO₂.
 2. The method of claim 1 wherein the mammal is a human.
 3. (canceled)
 4. The method of claim 1 wherein the tissue is affected by a medical condition.
 5. The method of claim 4 wherein the medical condition is selected from the group consisting of bedsores, wounds, burns, and ulcers.
 6. The method of claim 4 wherein the medical condition is a bacterial infection.
 7. The method of claim 6 wherein the bacterial infection is identified as an anaerobic pathogen bacterial infection.
 8. (canceled)
 9. (canceled)
 10. The method of claim 1 wherein the oxygen in the superoxygenated composition has a concentration from about 45 parts per million to about 220 parts per million.
 11. The method of claim 1 wherein the superoxygenated composition is at about 0° C. to about 34° C.
 12. (canceled)
 13. The method of claim 1 wherein the pharmaceutically acceptable vehicle comprises water.
 14. The method of claim wherein the oxygen microbubbles are between about 2μ and about 10μ in diameter.
 15. The method of claim 1 wherein the microbubbles are between about 0.6μ and about 5μ in diameter.
 16. The method of claim 1 wherein the superoxygenated composition is applied under agitation.
 17. The method of claim 16 wherein the agitation is provided in a whirlpool bath.
 18. The method of claim 1 wherein the pharmaceutically acceptable vehicle allows the superoxygenated composition to be administered in the form of a cream, lotion or gel.
 19. The method of claim 1 wherein the composition is applied by soaking, immersion, spraying, rubbing or aerosols. 